Animals and experimental set-up
All experiments were approved by the Institutional Animal Care and Use Committee of the VU University, and were conducted following the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes [10]. The performed research is in compliance with the modern ARRIVE guidelines on animal research [11].
Adult male Wistar rats (n = 16; body weight 262 ± 1 g; Harlan CBP, Horst, the Netherlands) were fed a high fat diet (HFD) for a period of 4 weeks (n = 8). Animals that received a diet low in fat and sugars (control diet; CD) for 4 weeks served as controls (n = 8). Animals were housed in a temperature-controlled room (20-23 °C; 40-60% humidity) under a 12/12 h light/dark cycle starting at 6.00 am. After 4 weeks, rats underwent an oral glucose tolerance test and (contrast) echocardiography during baseline and after dipyridamole infusion.
Diets
High fat diet was obtained from Research Diets (D12451, New Brunswick, NJ). The HFD consisted of 24 wt% protein, 24 wt% fat and 41 wt% carbohydrates (8.5 wt% starch, 20.1 wt% sucrose). Control diet (Teklad 2016) was obtained from Harlan (Horst, The Netherlands) and consisted of 17 wt% protein, 4 wt% fat and 61 wt% carbohydrates (45.1 wt% starch, 5.0 wt% sucrose).
Oral glucose tolerance test
Rats fasted overnight received an oral glucose load (2 g/kg of body weight). Blood glucose levels were measured from tail bleeds with a Precision Xceed Blood Glucose monitoring system (MediSense, UK) before (0) and 15, 30, 60, 90 and 120 min after glucose ingestion [12]. At similar time points, plasma insulin (LINCO research, St. Charles, Missouri) levels were measured as described previously [12].
Plasma measurements
Plasma haematocrit levels were determined using microcentrifugation. Plasma free fatty acids (WAKO NEFA-C, Wako Pure Chemical Industries, Osaka, Japan), plasma high-density lipoprotein (HDL) cholesterol and plasma low-density lipoprotein (LDL)/very-low-density lipoprotein (VLDL) cholesterol (Abcam, Cambridge, MA) were measured after overnight fasting as previously described [13, 14].
Cannulation of the jugular vein
For infusion of the contrast agent for echocardiography, a catheter was placed in the jugular vein under S-Ketamine (Ketanest®, 150 mg/kg, Pfizer, the Netherlands) and Diazepam (3 mg/kg, Centrafarm, the Netherlands) anaesthesia intraperitoneally. After surgery, echocardiography and contrast echocardiography to determine myocardial function and perfusion, respectively, were performed during baseline and dipyridamole-induced hyperaemia (20 mg/kg for 10 minutes) (Figure1).
Echocardiography
Echocardiography (Siemens, ACUSON, Sequoia 512) was performed as previously described [14]. Briefly, wall thickness (WT) and LV-dimensions (D) during end-systole (ES) and end-diastole (ED) were determined in the M- (motion) mode of the parasternal short-axis view at the level of the papillary muscles. Left ventricular contractile function was calculated by the fractional shortening = (EDD-ESD)/EDD·100%. Analyses were performed off-line (Image-Arena 2.9.1, TomTec Imaging Systems, Unterschleissheim/Munich, Germany). All parameters were averaged over at least three cardiac contractile cycles.
Myocardial contrast echocardiography
Contrast echocardiography was performed using a Siemens (ACUSON, Sequoia 512) equipped with a 14 MHz linear array transducer (Philips Healthcare, Best, The Netherlands). The contrast agent Sonovue® (Bracco Imaging, Italy), which contains 2x108-5x108/ml sulphur hexafluoride-filled, phospholipid-coated microbubbles with a diameter of 1–10 μm, was prepared according the manufacturer’s instructions and diluted twice by adding 5 ml NaCl (0.9%). Microbubbles were continuously infused into the jugular vein with a rate of 600 μl/min using a dedicated syringe pump (Vueject, Bracco SA, Switzerland). After two minutes of microbubble infusion, perfusion images were taken of the short axis view of the left ventricle at the level of the papillary muscles.
Low acoustic power (mechanical index [MI] 0.20 max) was used for microbubble detection. A perfusion sequence consisted of about 10 cardiac cycles of low MI imaging, followed by a burst of high acoustic power (MI 1.8) for complete contrast destruction. Subsequently, on average 20 cardiac cycles of low MI images were acquired at a frame rate of 14 Hz to allow contrast replenishment in the myocardium. All data were stored for offline analysis.
Myocardial contrast echocardiography analysis
Custom-designed software was used for analysis of the estimate of perfusion (Matlab, 7.10, R2010A, MathWorks Inc. Massachusetts, USA) with special thanks to R. Meijer. For each cardiac cycle the end-systolic frame was selected manually and regions of interest were drawn in the posterior wall in the short axis view of the left ventricle. Myocardial signal intensity extracted from the frames before microbubble destruction were used to calculate myocardial plateau intensity A. Myocardial signal intensities from the frames after microbubble destruction were corrected for background noise by subtracting the signal intensity of the first frame after microbubble destruction (Y0). These intensities were then fitted (Y = Y0 + (A-Y0)*(1-exp(−ß *x))) for calculation of ß, which was transformed into min-1 for further analysis (Figure2). The estimate of perfusion was calculated as A * ß. The flow reserve was calculated as the ratio of hyperaemic and baseline estimate of perfusion.
Statistical analysis
All data are presented as mean ± SEM. Between group comparisons (CD vs. HFD) were performed using a student t-test, whereas baseline vs. dipyridamole intervention was tested with a paired student t-test. Oral glucose tolerance test was tested with one-way ANOVA with repeated measurements and Bonferroni post-hoc test. p < 0.05 was considered as statistically significant.
